The present invention relates to methods for directing the evolution of microorganisms using mutator genes. Such methods provide a pool of microbial genetic diversity advantageous for industrial application, such as for the industrial production of heterologous proteins, such as hormones, growth factors and enzymes, and the biocatalytic production of chemicals, vitamins, amino acids and dyes.
The industrial applicability of microorganisms is restricted by their physiological limits set by solvent, pH, various solutes, salts and temperature. Organic solvents are generally toxic to microorganisms even at low concentrations. The toxicity of solvents significantly limits use of microorganisms in industrial biotechnology for production of specialty chemicals and for bioremediation applications. Solvent molecules incorporate into bacterial membranes and disrupt membrane structure (Isken and Bont, 1998, Extremophiles 2(3): 229-238); (Pinkart and White, 1997, J. Bacteriol. 179(13): 4219-4226); (Ramos, Duque et al., 1997, xe2x80x9dJ. Biol. Chem. 272(7): 3887-3890); (Alexandre, Rousseaux et al., 1994, FEMS Microbiol, Lett, 124(1): 17-22); and Kieboom, Dennis et al., 1998, J. of Bacteriology 180(24): 6769-6772). Classic strain improvement methods including UV and chemical mutagenesis have been applied for selection of more tolerant strains (Miller, J., xe2x80x9cA Short Course In Bacterial Genetics,xe2x80x9d Cold Spring Harbor Laboratory Press, 1992). A number of studies have been dedicated to identification and isolation of solvent tolerant mutants among various bacterial strains. Spontaneous E. coli solvent tolerant mutants and mutants isolated in the process of 1-methyl-3-nitrosoguanidine (NTG) mutagenesis were obtained from strain K-12 (Aono, Aibe et al., 1991 Agric. Biol. Chem 55(7): 1935-1938). The mutants could grow in the presence of diphenylether, n-hexane, propylbenzene, cyclohexane, n-pentane, p-xylene. Various Pseudomonas strains were able to adapt and to grow in a toluene-water two-phase system (Inoue and Horikoshi, 1989, Nature 338: 264-266), with p-xylene (Cruden, Wolfram et al., 1992, Appl. Environ. Microbiol. 58(9): 2723-2729), styrene and other organic solvents (Weber, Ooijkaas et al., 1993, Appl. Environ. Microbiol. 59(10): 3502-3504), (de Bont 1998, Trends in Biotechnology 16: 493-499). Yomano et al. isolated ethanol tolerant mutants which increased tolerance from 35 g/l to 50 g/l during 32 consequent transfers (Yomano, York et al., 1998, J. Ind. Microbiol. Biotechnol. 20(2): 132-138). High temperature evolution using E.coli has been disclosed in the art (Bennett, 1990, Nature, Vol. 346, 79-81) however the fitness gain was low as compared to the parent.
Strains of E. coli that carry mutations in one of the DNA repair pathways have been described which have a higher random mutation rate than that of typical wild type strains (see, Miller supra, pp.193-211). As reported by Degenen and Cox (J. Bacteriol., 1974, Vol. 117, No. 2, pp.477-487), an E. coli strain carrying a mutD5 allele demonstrates from 100 to 10,000 times the mutation rate of its wild type parent. Greener et al., xe2x80x9cStrategies In Molecular Biology,xe2x80x9d 1994, Vol. 7, pp.32-34, disclosed a mutator strain that produces on average one mutation per 2000 bp after growth for about 30 doublings.
Microorganisms are used industrially to produce desired proteins, such as hormones, growth factors and enzymes and to produce chemicals, such as glycerol and 1,3 propanediol (WO 98/21340 published May 22, 1998 and U.S. Pat. No. 5,686,276 issued Nov. 11, 1997), vitamins, such as ascorbic acid intermediates (1985, Science 230:144-149), amino acids, and dyes, such as indigo (U.S. Pat. No. 4,520,103, issued May 28, 1985). In spite of advances in the art, there remains a need to improve the microorganisms and methods for producing such desired proteins, chemicals, amino acids and dyes.
The present invention relates generally to methods for directing the evolution of a microorganism, that is for directing desired genetic change in a microorganism in response to conditions of selective pressure. In one aspect, the present invention relates to methods for evolving microorganisms to grow under extreme conditions, such as at high temperature, under conditions of pH extremes, in the presence of solvents, and in the presence of high salt. In another aspect, the present invention relates to methods for evolving a microorganism comprising at least one nucleic acid encoding a desired protein or an enzyme in an enzymatic pathway to grow under desired conditions.
The present invention is based, in part, upon the finding that microrganisms such as wild-type E.coli and E.blattae, can be evolved into microorganisms capable of growing in the presence of high solvents, such as DMF and 1,3 propanediol, using methods described herein. The present invention is also based, in part, upon the finding that E.coli can be evolved into a microorganism capable of growing at elevated temperatures using methods described herein. The present invention is further based, in part, upon the identification of the optimal mutation rate for a microorganism and the discovery that the mutation rate can be controlled.
Accordingly, the present invention provides a method for preparing an evolved microorganism comprising the steps of culturing a microorganism comprising at least one heterologous mutator gene, for at least 20 doublings under conditions suitable for selection of an evolved microorganism, wherein said heterologous mutator gene generates a mutation rate of at least about 5 fold to about 100,000 fold relative to wild type, and restoring said evolved microorganism to a wild type mutation rate. In one embodiment, the microorganism further comprises at least one introduced nucleic acid encoding a heterologous protein, said protein(s) including, but not limited to hormones, enzymes, growth factors. In another embodiment, the enzyme includes, but is not limited to hydrolases, such as protease, esterase, lipase, phenol oxidase, permease, amylase, pullulanase, cellulase, glucose isomerase, laccase and protein disulfide isomerase. The present invention encompasses genetic changes in the microorganism as well as changes in the introduced nucleic acid.
In yet a further embodiment, the microorganism further comprises introduced nucleic acid encoding at least one enzyme necessary for an enzymatic pathway. In one embodiment, the introduced nucleic acid is heterologous to the microorganism; in another, the introduced nucleic acid is homologous to the microorganism. In a further embodiment, the enzyme is a reductase or a dehydrogenase and said enzymatic pathway is for the production of ascorbic acid or ascorbic acid intermediates. In an additional embodiment, the enzyme is glycerol dehydratase or 1,3-propanediol dehydrogenase and said enzymatic pathway is for the production of 1,3 propanediol, 1,3 propanediol precursors or 1,3 propanediol derivatives. In another embodiment, the enzyme is glycerol-3-phosphate dehydrogenase or glycerol-3-phosphate phosphatase and said pathway is for the production of glycerol and glycerol derivatives. In a further embodiment, the enzymatic pathway is for the production of amino acids, such as tryptophane or lysine or dyes, such as indigo.
In one embodiment of the present invention, the microorganism is cultured for between about 20 to about 100 doublings; in another embodiment, the microorganism is cultured for between about 100 to about 500 doublings; in yet another embodiment, the microorganism is cultured for between about 500 to about 2000 doublings and in a further embodiment, the microorganism is cultured for greater than 2000 doublings. In one embodiment, the mutator gene generates a mutation rate of at least about 5 fold to about 10,000 fold relative to wild type; in another embodiment, the mutator gene generates a mutation rate of a least about 5 fold to about 1000 fold and in another embodiment, the mutator gene generates a mutation rate of about 5 fold to about 100 fold over wild type.
In one embodiment, an evolved microorganism comprises from about 3 to about 1000 selected mutations in about 3 to about 500 genes and may further comprises from about 20 to about 100,000 neutral mutations. In one aspect, the mutations generated are non-specific and in another aspect, the mutations generated are specific.
In one embodiment of the present invention, the microorganism comprises a plasmid comprising the heterologous mutator gene and said step of restoring said evolved microorganism to a wild type mutation rate comprises curing the evolved microorganism of said plasmid. In another embodiment, the plasmid comprises a temperature sensitive origin of replication and the curing comprises growing the evolved microorganism at a restrictive temperature. In a further embodiment, the microorganism comprises at least one copy of the mutator gene in the chromosome and said step of restoring said evolved microorganism to a wild type mutation rate comprises excision or removal of said mutator gene from the host genome or the replacement of the mutator gene with a functional (non-mutator) allele of the same gene.
In one embodiment, the present invention comprises the use of at least one mutator gene to evolve a microorganism. In another embodiment, the mutator gene includes but is not limited to a mutD gene mutation, a mutT gene mutation, a mutY gene mutation, a mutM gene mutation, a mutH gene mutation, a mutL gene mutation, a mutS gene mutation or a mutU gene mutation and homologues of these DNA repair genes which have been mutated as long as the mutated gene has impaired proofreading function. In a further embodiment, the mutator gene comprises at least one of the mutD mutations disclosed herein in Table 1.
In one embodiment of the present invention, conditions suitable for selection include but are not limited to culturing said microorganism in the presence of at least one organic solvent, such as for example, alcohols, diols, hydrocarbon, mineral oil, mineral oil derived products, halogenated compounds and aromatic compounds; in the presence of high temperature, such as in the range of 42xc2x0-48xc2x0 C.; in the presence of high salt, and in the presence of extreme pH conditions, such as alkaline or acidic conditions.
The present invention encompasses methods for evolving gram positive and gram negative microorganisms as well as yeast, fungus and eucaryotic cells including hybridomas. In one embodiment, the gram negative microorganism includes members of Enterobacteriaceae and in another embodiment comprises Eschericia and in another embodiment comprises E.coli and E.blatte. 
In further embodiments of the present invention, the evolved microorganism includes E.coli having ATCC accession number PTA-91 and E.blattae having ATCC accession number PTA-92.
The present invention also provides expression vectors and host cells comprising a mutator gene and methods for producing such vectors and host cells.